(a) Field of the Invention
This invention relates to methods for treating fractionated patterns of serums formed by electrophoresis and more particularly to a method of determining respective boundary points of a fractionated pattern.
(b) Description of the Prior Art
FIG. 1 shows a basic pattern of concentration distribution on fractionated patterns formed by electrically energizing with an electrophoretic apparatus a carrier made of cellulose acetate film onto which man's serum is applied (a healthy man's serum generally shows this pattern). Such an electrophoretic patterns usually consists of five fractions of A, B, C, D and E including five peaks of a.sub.0, a.sub.1, a.sub.2, a.sub.3 and a.sub.4 corresponding to albumin (A), .alpha..sub.1 globulin (B), .alpha..sub.2 globulin (C), .beta. globulin (D), and .gamma. globulin (E) respectively. Diagnosis or distinguishment between normality and abnormality is done on the basis of an analog diagram and values in percentage of the respective fractions. However, patterns of concentration distribution on fractionated patterns of actual sample to be examined may include peaks produced by various cases in addition to those shown in FIG. 1. The pattern illustrated in FIG. 2, for example, includes a peak designated as a.sub.5 in addition to the five peaks mentioned above. This peak is produced due to turbidity in serum which allows a substance insensible of electrophoresis to remain at the position of sample application. The electrophoretic pattern shown in FIG. 3 includes an additional peak at the position of a.sub.6, whereas the one shown in FIG. 4 includes an additional peak at the position of a.sub.7. These peaks are produced by fractionation of certain components contained in the sera depending on their freshness, the additional peak shown in FIG. 3 being produced by .beta. lipoprotein and that in FIG. 4 being produced by .beta..sub.1c globulin.
When colorimetry is done on a sample which shows peaks in addition to the five basic peaks, inconvenience is caused in automatic processing with a computer of data obtained by colorimetry. FIG. 5 shows an example of configuration of a densitometer and a photometric apparatus which are currently employed. In the block diagram shown in FIG. 5, the light emitted from a light source lamp 1 is passed through a lens 2, a filter 3 and a slit 4, used for irradiating a carrier 5 (described later) and detected with a photo detector element 7. The carrier 6 has fractionated patterns 6, 6', 6",--of sera formed thereon as shown in FIG. 6, and is placed between the light source and the detector for photometry of the individual fractionated patterns 6, 6', 6",--while scanning in the direction perpendicular to the shifting direction of the carrier 5. That is to say, the light emitted from the light source lamp 1 and passing through the sample (fractionated pattern or a serum) is received by the photo detector element 7, whose output corresponding to sample concentration is amplified with a preamplifier 8, converted by a logarithmic converter 9 into a logarithmic value and used for preparing an analog densitogram as shown in FIG. 1. Successively, output from the logarithmic converter 9 is inputted into an A/D converter 10 and converted into a digital signal by operating a conversion command signal generator 11 with a photometry command 11a from a computer 12. Value in percentage of each fraction is determined on the basis of the digital data obtained at this stage.
For the operations described above, it is sufficient to determine points of local minimum values as boundary points in such as case as shown in FIG. 1. In cases of the electrophoretic patterns divided into more than five fractions as illustrated in FIG. 2 through FIG. 4, however, it is impossible to determine values of the five fractions. In a case where an electrophoretic pattern has more than five fractions, it is therefore required for the analyst to check an analog pattern and electrophoretic pattern for recalculation through processing to attribute the additional peaks to any one of the areas corresponding to albumin, .alpha..sub.1 globulin, .alpha..sub.2 globulin and .gamma. globulin. In case of abnormal fractions due to disease, they may be reported with no attempt made for data processing.
A treating method wherein, under such circumstances as are described above, even in case an electrophoretic pattern is fractionated into six or more fractions, it will be able to be automatically rearranged as of normal five fractions by a computer is suggested by U.S. Pat. No. 4,295,949. This method (which shall be called the first conventional method hereinafter) shall be briefly explained as follows.
First, such serum to be a standard as is marketed is electrophoresizied as a control serum and is further measured with a densitometer to obtain an electrophoretic pattern of five fractions. The positions and boundary points of respective peaks of the electrophoretic pattern will be substantially determined by the kind of the carrier and the electrophoresizing conditions. Therefore, if the kind of the carrier and the electrophoresizing conditions are the same, the electrophoretic pattern of a serum to be tested will not be substantially different from that of the standard or normal serum. The standard lengths (from the basic point to the respective peak tops or boundary points) in the electrophoretic pattern of this standard serum are determined as follows. That is to say, such electrophoretic pattern of the standard serum as in FIG. 7 obtained as described above is sampled at fixed time intervals and is A/D-converted and the concentrations at the respective sampling points are memorized. In considering the axes x and y as shown in FIG. 7, it is found that the values of y at the respective sampling points on the axis x correspond to the concentrations (digital values) at the respective sampling points. On the basis of the concentrations at these respective sampling points, the detection of the respective boundary points shall be explained. As the respective boundary points are points on the axis x corresponding to the respective valley bottoms of the electrophoretic pattern, if any sampling point on the axis x is x.sub.b and the value of y at this sampling point is y.sub.b and, if the value of y at a sampling point x.sub.b-1 is y.sub.b-1 and the value of y at a sampling point x.sub.b+1 is y.sub.b+1, a sampling point x.sub.b having such values of y.sub.b as will satisfy the relations of EQU y.sub.b &lt;y.sub.b-1 and y.sub.b &lt;y.sub.b+1
will be a boundary point. Next, the positions of peaks shall be described. There are respectively a peak top a.sub.0 between the basic point x.sub.0 and boundary point b.sub.1, a peak top a.sub.1 between the boundary points b.sub.1 and b.sub.2, a peak top a.sub.2 between the boundary points b.sub.2 and b.sub.3, a peak top a.sub.3 between the boundary points b.sub.3 and b.sub.4 and a peak top a.sub.4 between the boundary point b.sub.4 and end point x.sub.n. These respective points satisfy the relations of y.sub.a &gt;y.sub.a-1 and y.sub.a &gt;y.sub.a+1. The positions of the thus determined points b.sub.1, b.sub.2,--and a.sub.0, a.sub.1,--are in proportional relations with the lengths on the axis x from the basic point to the respective points and correspond to 1:1. Therefore, these coordinates (in the fixed time intervals) may be used instead of the lengths from the basic point.
As in the above, the fractionation of the standard serum ends.
Then, boundary points are determined by using the above described method on a pattern obtained by electrophoresizing a sample to be measured. Then, with reference to the points a.sub.0, a.sub.1, a.sub.2 and a.sub.3 on the axis x corresponding to the respective peak tops in the electrophoretic pattern of the standard serum, these respective points are positioned on the axis x of the electrophoretic pattern of the sample to be measured as shown in FIG. 8. In the electrophoretic pattern of this sample to be measured, in case the number of the boundary points located in each of the sections between the points a.sub.0 and a.sub.1, between a.sub.1 and a.sub.2, between a.sub.2 and a.sub.3 and between a.sub.3 and a.sub.4 is counted and is 1, it will be made a boundary point. In case the number is 2 or more, the point of the smallest concentration or value of y will be made a boundary point and the others will be canceled. For example, in FIG. 8, as there is one boundary point (b.sub.1 and b.sub.3) in each of the sections between the points a.sub.0 and a.sub.1 and between the points a.sub.2 and a.sub.3, they will be respectively made the first boundary point and third boundary point, as there are three boundary points indicated by b.sub.5, b.sub.2 and b.sub.6 between the points a.sub.1 and a.sub.2, the point b.sub.2 having the smallest value among them will be made the second boundary point and, as there are two boundary points b.sub.7 and b.sub.4 between the points a.sub.3 and a.sub.4, the point b.sub.4 of them will be made the fourth boundary point. The boundary point (b.sub.8 in FIG. 8) further following the point a.sub.4 will be all canceled. That is to say, the boundary points by .beta.-lipoprotein, .beta..sub.1c -protein and impurities always take values higher than those of normal five boundary points. Therefore, according to the above mentioned treating method, it is possible to make five boundary points.
The basic point is made the origin of the coordinates in the above explanation but the point a.sub.0 corresponding to the first peak may be made the origin of the coordinates. However, in this first conventional method, a standard serum to be always normally fractionated is required. Some marketed standard serums are not always normally fractionated. It is thus difficult to select a standard serum. The sample to be examined will be influenced by the conditions of the place in which it is kept and will not be fractionated correctly into five fractions when it is polluted with bacteria. Therefore, it is a very difficult problem to keep a standard serum.
In order to solve such problem, there is recently suggested a fractionating method in electrophoresis wherein correct boundary positions are determined by utilizing the above described first conventional method and event without using a standard serum.
This method (which shall be called the second conventional method herein-after) shall be explained in the following. FIG. 9 is a block diagram of a fractionating apparatus by utilizing the first method. An electrophoretic pattern of a standard serum is measured in a measuring device 13, the measured values are fractionated and judged by the above described method in a boundary position judging device 14 and the values (a.sub.0, a.sub.1, a.sub.2, a.sub.3 and a.sub.4) of the coordinates x of respective peak points and the boundary positions (b.sub.1, b.sub.2, b.sub.3 and b.sub.4) are sent to and memorized in a basic position memorizing device 15. Then, an electrophoretic pattern of a sample to be examined is measured in the measuring device 13 and the values of the coordinates x corresponding to respective maximum values and minimum values are determined in the boundary position judging device 14 from the measured results. The thus determined values are compared with the values of the standard serum memorized in advance by a five-fraction treating device 16, correct boundary positions are determined by the already explained method and fractionated data are obtained on the basis of this fractionated data output.
In the second conventional method, the basic position is determined by the normally five-fractionated data of the sample to be examined without using a standard serum as in the first conventional method and is referred to in fractionating the sample to be measured into five fractions. It is known that the integrated values of the concentrations of the respective fractions, for example, of a normal human serum are in a fixed range. In the second conventional method, by noting this point, whether the fractions are normal or not is determined by whether the sample to be examined has five fractions and further the above mentioned integrated value of the concentration or the ratio of one fraction to the other fraction is in a fixed range or not, the basic position is determined on the basis of the fractions judged to be normal and the boundary positions of the sample to be examined are determined with reference to it.
Now, the second conventional method shall be explained on the basis of the block diagram in FIG. 10. The measured values of the sample to be examined are determined in the measuring device 13, the values of the coordinates x of the maximum values and minimum values of them are determined in the boundary position judging device 14 the same as in the case of FIG. 9. Then, whether the values determined in the boundary position judging device 14 have normal five fractions or not is judged in a normal fraction judging device 17. They have five fractions, the integrated values of the concentrations of the respective fractions fractionated by the respective valley bottom positions (b.sub.1, b.sub.2, b.sub.3 and b.sub.4) are determined and whether the fractions are normal or not is judged in the method by whether these integrated values are in the range set in advance or not. Only the data of the maximum values and minimum values judged to be normal in this normal fraction judging device 17 are sent to a next basic position calculating device 18 and a proper basic position is calculated by a proper statistical treatment. The basic position calculated here is sent to and memorized in a basic position memorizing device 19. The basic position memorized here corresponds to the basic position of the standard serum memorized in the basic position memorizing device 15 in the first conventional method (FIG. 9). Then the same as in the method of FIG. 9, the data from the boundary position judging device 14 of the sample to be examined are compared with the basic position memorized in the basic position memorizing device 19 and sent to the five-fraction treating device 16 through a switching device 20 and data fractionated with correct fractionated values are put out of the five-fraction treating device 16 and the integrated values of the concentrations of the respective fractions are calculated. Thus the basic position is determined by using the sample itself to be examined but without using the standard serum and is used to fractionate electrophoretic patterns of other samples to be examined. However, in the case of this second conventional method, though there is an advantage that it is not always necessary to use a standard serum, it has been still insufficient to properly fractionate the electrophoretic pattern of a sample to be examined into five fractions.